Since the demonstration by our laboratory over six years ago that insulin receptors in cultured endothelial cells are able to transport insulin from apical to basolateral surface without significant degradation, strong physiological evidence has supported this idea that insulin receptor on the endothelium can affect insulin action by regulating the delivery of insulin to its target cells. Studies over the last few years have shown that insulin receptor number, transport, and endosomal acidification can affect the transcytosis process. One important factor that will modulate insulin receptor trafficking in endothelial cells appears to be phorbol ester (PMA), an activator of protein kinase C (PKC). In this proposal, we will e focusing our studies on the role of PKC in the regulation of receptor-mediated transcytosis of insulin across endothelial and epithelial cells. We will determine the role of PKC mediated serine/threonine phosphorylation in regulating insulin receptor trafficking and insulin transcytosis by a) testing the effect of PKC activators and inhibitors; b) the determination of the site on insulin receptor which are phosphorylated by PMA in cells or by purified PKC in vitro; c) developing novel site-specific IR mutants of PKC phosphorylation sites; d) the characterization of NRK and endothelial cells transfected with deletional and site-specific mutants of insulin receptors. The second aim is to characterize the vesicular pathways used by insulin and insulin receptors in endothelial cells by kinetic studies using a photoactivatable insulin analog (125I-BPA-INS) and subcellular fractionation. In addition, internalization, processing, internalization and transcytosis of insulin will be studied in capillary endothelial cells from diabetic rats, since information has been presented indicating that the structure and number of IR in these cells are altered. These studies will extend our knowledge on the signals for receptor-mediated transcytosis of insulin across endothelial and epithelial cells to the molecular and biochemical level. In addition, studies using cells from diabetic rats could provide suggestive evidence of a role for transcytosis in causing insulin resistance.